Role of perfusion in the generation of stromal vascular fraction-based patches with angiogenic potential

Restoring of the coronary flow upon thrombotic occlusion of a coronary artery is fundamental to prevent progression of the infarct size and a global loss of cardiac function, ultimately leading to end-stage heart failure condition. There is an open clinical need for the development of novel angiogenic therapies capable to promote ingrowth of vessels at the infarction border area and, possibly, the consequent rescue of dysfunctional cardiomyocytes in patients suffering from post-myocardial infarction chronic ischemia. Adipose-derived stromal vascular fraction (SVF) cells are of particular relevance when thinking of a cell-based approach. 3D patches offer a promising strategy to increase survival and repair potential upon implantation in a hostile environment such as the infarcted myocardium. Priming of 3D SVF-patches by perfusion culture has been shown to increase their engraftment and angiogenic potential upon in vivo subcutaneous implantation, orchestrated by a perfusion-dependent in vitro enrichment of a pericyte-like SVF subpopulation defined as CD45- CD146+ CD34-, and sustained by the presence of a microenvironment containing extracellular matrix (ECM) components.
In this study, we aimed at: i) depicting the possible mechanisms responsible for the enrichment of CD45- CD146+ CD34- cells in perfusion-cultured SVF and ii) evaluating the engraftment and vascularization potential of CD45- CD146+ CD34--enriched SVF patches upon implantation in a ‘proof-of-concept’ model in healthy nude rat hearts.
Enrichment of CD45- CD146+ CD34- cells was found to follow a flow-rate dependent pattern, as well as the deposition of ECM proteins such as fibronectin and laminin. The possible mechanisms behind these observations were evaluated by hypothesizing ERK1/2 pathway to have a major role, as it was activated under perfusion culture. Surprisingly, inhibition of ERK1/2 pathway under perfusion impaired the enrichment of CD45- CD146+ CD34- cells, while the other SVF subpopulations remained unchanged. These results proposed perfusion-dependent activation of ERK1/2 pathway to be key in promoting the maintenance and enhancement of CD45- CD146+ CD34- cells. Whether ERK1/2 was exclusively active on this subpopulation, or it contributed in the generation of a microenvironment supporting their enrichment by also acting on the other SVF components, still remains to be elucidated.
Perfusion-based SVF patches, enriched in CD45- CD146+ CD34- cells and ECM components, showed significantly higher levels of engraftment upon epicardial implantation in healthy hearts. Interestingly, SVF cells primed under perfusion culture promoted an increased vascularization potential, not only within the patch itself (intrinsic) but also around its border with the healthy myocardium (extrinsic).
In conclusion, this work provides a deep characterization at the cellular and ECM level of perfusion-cultured 3D SVF patches. Further studies should be designed to understand a possible mechanosensitive-dependent enrichment of CD45- CD146+ CD34- cells. Moreover, the observations from the in vivo implantations set the bases for a future evaluation of 3D SVF perfusion-cultured patches in a diseased model of chronic cardiac ischemia.